The present invention relates to compositions and methods for transferring nucleic acids into cells in vitro and in vivo. The compositions comprise a transfection reagent and one or more detergents. In preferred embodiments, the compositions comprise delivery systems providing nucleic acid transfer complexes that transfect cells with high efficiency.
The efficient delivery of biologically active compounds to the intracellular space of cells has been accomplished by the use of a wide variety of vesicles. One particular type of vesicle, liposomes, is one of the most developed types of vesicles for drug delivery. Liposomes are microscopic vesicles that comprise amphipathic molecules that contain both hydrophobic and hydrophilic regions. Liposomes can contain an aqueous volume that is entirely enclosed by a membrane composed of amphipathic molecules (usually phospholipids).
Liposome drug carriers have been under development since the 1970""s. Liposomes are formed from one to several different types of amphipathic molecules. Several methods have also been developed to complex biologically active compounds with liposomes. For example, a biologically active compound can be entrapped within the internal aqueous phase, within the lipid phase, or complexed to the outside of the liposome.
Liposomes can be divided into three groups based upon their overall size and lamellar structure. Small uni-lamellar vesicles (SUV), which are typically prepared by sonication, are 20 to 30 nm in diameter and contain one single lipid bilayer surrounding the aqueous compartment. Multi-lamellar vesicles (MLV) are prepared by simply mixing amphipathic molecules in an aqueous phase and contain multiple aqueous compartments and bilayers. Large uni-lamellar vesicles (LUV) are most commonly prepared by reverse-phase evaporation. After subsequent pore filtration, LUV""s are usually 150 to 200 nm in diameter.
Liposomes can also be classified according to mechanisms by which they attach to a target cell. Gangliosides, polysacharrides and polymers such as polyethylene glycol have been attached to liposomes (termed xe2x80x9cStealth Liposomesxe2x80x9d) to decrease their non-specific uptake by the reticuloendothelial system in vivo. Antibodies, polysaccharides, sugars, and other ligands have been attached to liposomes to enable the tissue and cell specific delivery of biologically active compounds. Other cellular and viral proteins have also been incorporated into liposomes for targeting purposes and for their fusogenic properties.
Liposomes typically deliver a biologically active compound found within their aqueous space to target cells by fusing with either the plasma membrane or an internal membrane of the cell after endocytosis of the liposome. Fusion of the liposome membrane with the cellular membrane is one of the critical steps in the efficient delivery of substances to the cell. Certain types of liposomes are endocytosed by certain types of cells. If a liposome is endocytosed by a receptor-mediated pathway, then it enters an endosome. In order for the biologically active compound contained within or associated with the liposome to reach its target sites and receptors, it is essential that the compound escape or be released from the endosome and avoid degradation in the lysosomes.
Efficient nucleic acid transfer in vitro has been accomplished with the use of positively-charged liposomes that contain cationic lipids. For example, the cationic lipid, N-1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) was the first cationic lipid used for DNA transfections. DOTMA was combined with dioleoylphosphatidylethanolamine (DOPE) to form liposomes that spontaneously complexed with nucleic acids (DNA and RNA) and provided relatively efficient transfections. Other neutral lipids have been used in conjunction with amphipathic compounds to form liposomes. These have generally been chosen from the group consisting of phosphatidylethanolamines (e.g., DOPE), phosphatidylcholines, or phosphatidylserines, wherein the acyl group chain length is between 16 and 20. Another compound used to form liposomes suitable for transfecting nucleic acids into cells is cholesterol.
These liposomes are simply mixed with the nucleic acid and then applied to the cells in culture. Complete entrapment of the DNA or RNA molecules occurs because the positively-charged liposomes naturally complex with negatively-charged nucleic acids. DNA has been shown to induce fusion of cationic liposomes containing DOTMA/DOPE. The procedure with the cationic lipids is generally as or more efficient than the commonly-used procedure involving the co-precipitation of calcium phosphate and DNA.
DOTMA/DOPE liposomes have, however, substantial cytotoxicity, particularly in vivo. A variety of cationic lipids have been made in which a glycerol or cholesterol hydrophobic moiety is linked to a cationic headgroup by metabolically degradable ester bond. These have included 1,2-bis(oleoyloxy)-3-(4xe2x80x2-trimethylammonio)propane (DOTAP), 1,2-dioleoyl-3-(4xe2x80x2-trimethylammonio)butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) and cholesteryl (4xe2x80x2-trimethylammonio)butanoate (ChoTB). However, there is no evidence of reduced cytotoxicity in comparison of these ester bond-containing cationic lipids as compared to DOTMA. Stearylamine, a cationic lipid has been used in liposomes but it had great cytotoxicity and was not been reported to mediate DNA transfer. Another detergent, cetyltrimethylammonium bromide (CTAB), when combined with DOPE, was able to mediate DNA transfection, but it had significant cytotoxicity. A series of cationic, non-pH sensitive lipids that included DORI (1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide), DORIE (1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide), and DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) have been reported and studied. Other non-pH-sensitive, cationic lipids include: O,Oxe2x80x2-didodecyl-N-p-(2-trimethylammonioethyloxy)benzoyl-N,N,N-trimethylam monium chloride, Lipospermine, DC-Chol (3xc3xa1-N-(Nxe2x80x2,Nxe2x80x3-dimethylaminoethane)carbonylcholesterol), lipopoly(L-lysine), cationic multilamellar liposomes containing N-(xc3xa1-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), TRANSFECTACE (1:2.5 (w:w) ratio of DDAB which is dimethyl dioctadecylammonium bromide and DOPE) (Life Technologies) and LIPOFECTAMINE (3:1 (w:w) ratio of DOSPA which is 2,3-dioleyloxy-N-20({2,5-bis (3-aminopropyl)amino-1-oxypentyl}amino)ethyl-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminium trifluoroacetate and DOPE) (Life Technologies). While much development has occurred, the art is still in need of improved transfection compositions and methods for providing efficient, low toxicity delivery of agents in vitro and in vivo.
Furthermore, most transfection reagents have been optimized to transfect mammalian cells. In vitro, these cells are generally maintained at 37xc2x0 C. Only two commercially available transfection reagents are claimed to be most efficient for the transfection of insect cells, which are maintained at a temperature of 25-27xc2x0 C. Studies on cell membrane composition demonstrated that there are significant composition differences between mammalian and insect cells. Thus, the art is also in need of efficient, low toxicity delivery systems for the delivery of agents to cells other than mammalian cells or to cells under non-standard growth conditions.
The present invention relates to compositions and methods for transferring nucleic acids into cells in vitro and in vivo. The compositions comprise a transfection reagent and one or more detergents. In preferred embodiments, the compositions comprise one or more amphipathic compounds and one or more neutral lipids. In preferred embodiments, the compositions comprise delivery systems providing nucleic acid transfer complexes that transfect cells with high efficiency. The transfection complexes of the present invention find use, for example, in the high-efficiency transfection of cells. A variety of cells may be transfected with high efficiency using the compositions and methods of the present invention, including, but not limited to, insect cells and keratinocytes. As demonstrated by the data herein, the transfection complexes of the present invention provides substantially higher transfection efficiencies than available systems.
In some embodiments of the present invention, the transfection complex comprises an amphipathic compound, a neutral lipid, and a detergent. In some preferred embodiments, the detergent comprises a short lipid chain (e.g., dilauroyl phosphatidylethanolamine [DLPE]). Such complexes significantly alter the efficiency for transferring nucleic acids into cell types that are maintained in non-traditional conditions (i.e., non-mammalian cell types maintained, for example, at temperature other than 37xc2x0 C.), as well as cells that are maintained under standard or traditional conditions. For example, in some preferred embodiments, the transfection complex comprises the amphipathic compound DOTAP, which is mixed with the neutral lipid DOPE, and the detergent DLPE. Although any suitable combination of the components may be used (e.g., any combination that permits transfection of cells), in particularly preferred embodiments, the components are provided in a ratio of 10:9:1 (DOTAP:DOPE:DLPE, w:w:w), respectively.
In some embodiments of the present invention, the transfection complex comprises an amphipatic compound and a nucleic acid-binding protein. Preferably, the DNA-binding protein is cationic (net positive charge) such as a histone protein. Hi histone protein is the preferred histone type. In yet another preferred embodiment, histone Hi protein is used as a DNA-binding protein and 1,4-bis(3-oleoylamido propyl)piperazine is used for the amphipathic compound. In preferred embodiments, a detergent is added to the transfection reagent mixture (e.g., DLPE or CHAPS).
In some embodiments of the present invention, the transfection complex comprises an amphipathic compound, a cationic protein, and a neutral lipid. Such complexes significantly alter the efficiency for transferring nucleic acids into certain cell types. For example, in some preferred embodiments, the transfection complex comprises the amphipathic compound 1,4-bis(3-oleoylamidopropyl) piperazine, the protein histone H1, and the neutral lipid DLPE. In particularly preferred embodiments, the components are provided in a ratio of 1:3:0.125 (w:w:w).
In some embodiments of the present invention, the transfection complex comprises an amphipathic compound, a cationic protein, and a detergent. In some preferred embodiments, the detergent comprises 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Such complexes significantly alter the efficiency for transferring nucleic acids into certain cell types. For example, in some preferred embodiments, the transfection complex comprises the amphipathic compound 1,4-bis(3-oleoylamidopropyl) piperazine, the protein histone H1, and the detergent CHAPS. In particularly preferred embodiments, the components are provided in a ratio of 1:3:0.25 (w:w:w).
In some embodiments, the present invention provides a process of delivering a biologically active substance to a cell comprising exposing the cell to the biologically active substance in the presence of a delivery system of the present invention. In a preferred embodiment, the biologically active substance is a nucleic acid. In preferred embodiments, the delivery system comprises liposomes that are complexed with the nucleic acid.
In preferred embodiments, the present invention provides a process for delivering nucleic acids into insect cells. While the present invention is not limited by the nature of the insect cells, in preferred embodiments the insect cells are Sf9, Sf21, High Five, or S2 cells.
In preferred embodiments, the present invention provides a process for delivering nucleic acids into keratinocytes. While the present invention is not limited by the nature of the keratinocytes, in preferred embodiments the keratinocytes are NIKS (Normal Immortalized Keratinocyte that produce Skin).
In preferred embodiments, the present invention provides a process for delivering nucleic acids into 293 cells.
The present invention further provides compositions comprising a transfection complex (e.g., liposome), wherein the transfection complex comprises a detergent. For example, in some embodiments, the transfection complex comprises amphipathic compounds. In other preferred embodiments, the transfection complex is capable of complexing nucleic acids. In preferred embodiments, the detergent comprises lipids (e.g., phospholipids) with acyl chains shorter than 18 carbons, more preferably shorter than 16 carbons or less (e.g., 15, 14, 13, 12, . . . ). In yet other preferred embodiments, the detergent has a critical micelle concentration equal to or higher than dipalmitoylphosphatidylcholine (DPPC).
The present invention also provides a composition for transfecting cells comprising an amphipatic compound; a lipid, said lipid comprising phospholipid, glycolipid, sphingosin-containing lipid, cholesterol or combinations thereof, said phospholipid, glycolipid, and sphingosin-containing lipid comprising an acyl group chain length equal to or longer than 16 carbons; and a detergent. While the present invention is not limited to any particular ratio of the consitituents, in preferred embodiments, the molar ratio of the amphipathic compound to the lipids is from 10:1 to 1:10. In preferred embodiments, the detergent comprises a neutral lipid with an acyl chain length equal to or smaller than 16 carbons. While the present invention is not limited to any particular ratio of the consitituents, in preferred embodiments, the molar ratio of the lipid to the detergent is from 100:1 to 1:100. Suitable detergents for use with the present invention include, but are not limited to, dilauroylphosphatidylethanolamine (DLPE), dilauroylphosphatidylcholine (DLPC), and 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).
In some preferred embodiments, the present invention further provides a composition comprising 1,2-bis(oleoyloxy)-3-(4xe2x80x2-trimethylammonio)propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), and DLPE. While the present invention is not limited to any particular ratio of the consitituents, in preferred embodiments, the molar ratio of DOTAP to phospolipids is appoximately 1:1 and wherein the ratio of DOPE to DLPE is 100:1 to 1:100. For example, is some embodiments, the ratio of DOPE to DLPE is approximately 19:1 or 9:1.
The present invention also provides a composition comprising DOTAP, DOPE, and DLPC wherein the ratio of DOTAP to neutral lipids is approximately 1:1 and wherein the ratio of DOPE to DLPC is 100:1 to 1:100. For example, in some embodiments, the ratio of DOPE to DLPC is approximately 19:1 or 9:1.
The present invention further provides a composition comprising: amphipathic compounds capable of complexing nucleic acids; cationic polymers; and a lipid selected from the group consisting of phospholipids, glycolipids, cholesterol, and sphingosin-containing lipids, wherein the acyl group chain length of said lipid is smaller than 16 carbons; wherein the molar ratio of the amphipathic compounds to the lipids is 100:1 to 1:100. In preferred embodiments, the lipid is dilauroylphosphatidylethanolamine (DLPE) or 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).
The present invention also provides a composition comprising 1,4-bis(3-oleoylamidopropyl) piperazine, histone H1, and detergent. In some preferred embodiments, the ratio of 1,4-bis(3-oleoylamidopropyl) piperazine to histone H1 is 3 to 1, and the ratio of 1,4-bis(3-oleoylamidopropyl) piperazine to detergent is 100:1 to 1:100. In some preferred embodiments, the detergent is DLPE or CHAPS. In particularly preferred embodiments, the ratio of 1,4-bis(3-oleoylamidopropyl) piperazine to histone H1to detergent is 3:1:0.125.
The present invention further provides kits containing any of the transfection complexes described herein. Such kits may further comprise cells for transfection, instructions, and control materials. In some preferred embodiments, the kits comprise a transfection complex, said transfection complex comprising a detergent. For example, in some embodiments, the detergent comprises phospholipids with acyl chains shorter than 16 carbons.